1. Field of the Invention
The present invention relates to a method and apparatus for the separation and analysis by liquid chromatography. More particularly, it relates to a method and apparatus for the separation and analysis by liquid chromatography, whereby a variety of substances can be separated and purified quickly and highly efficiently, and can be analyzed with high sensitivity.
2. Description of the Related Art
In recent years, there has been a rapid progress in the techniques for the separation and analysis by liquid chromatography, and it has been made possible to separate various substances ranging from low molecular weight to high molecular weight substances. However, in general, in aqueous liquid chromatography, the eluting solution (mobile phase) contains various salts in high concentrations depending upon the sample to be eluted. Therefore, there have been problems such that in the analysis of the eluted sample, the detection by an electric conductance meter, an ultraviolet absorption meter or a differential refractometer is difficult, and that in the fractionation of the sample into the individual components, desalination of the eluted sample is problematic. For the desalination of such a sample, it is conceivable to employ diffusion dialysis by means of a cellulose-type dialysis membrane, ultrafiltration by means of an ultrafilter and electrodialysis. However, in the diffusion dialysis or in the ultrafiltration, leakage of a sample having a molecular weight of 5000 or less is substantial, and no adequate desalination can be accomplished. On the other hand, in the electrodialysis, the apparatus is complex and large-sized, and as such, it is not suitable for application to chromatography. Besides, it is difficult to thereby accomplish the desalination to a low salt concentration (a few hundreds ppm or less). Further, in the separation by liquid chromatography, it becomes necessary to introduce a certain electrolyte to the column eluate, for instance, for the exchange of the buffer solution after the elution from the column for the stabilization of the sample. In such a case, the conventional methods have the same difficulties as mentioned above when the molecular weight of the sample is low.
In liquid chromatography, particularly in ion chromatography, it has been proposed to improve the analytical sensitivity by treating the eluate from the column by a membrane (e.g. Japanese Unexamined Patent Publication No. 66052/1983 and European Pat. No. 32770). However, in this case, a cation exchange membrane is used between the column eluate and the regenerant. Therefore, although the cation exchange is adequately conducted, other ionic impurities contained in the column eluate can not be removed, and no adequate improvement in the sensitivity is accomplished. Further, it is thereby difficult to introduce the necessary electrolyte to the column eluate.